The present invention relates to methods, devices and systems for performing digital measurements. More specifically, the present invention relates to methods, devices and systems for performing digital measurements in varying volumes.
Digital measurements are becoming increasingly more important in biology, owing to their robustness, higher sensitivity and higher accuracy that they offer. In addition, unlike analog measurements, where the measurement often must be calibrated with a running standard, digital measurements, based on counting of binary yes or no responses, do not require calibration and thus save user time, enhance robustness and ease of the assay.
An important application for digital assays is the accurate quantification of DNA or RNA that is present in a sample. Here, the most widely used method to detect DNA or RNA is the polymerase chain reaction (PCR), where the sample is usually cycled between two or three temperatures around 60° C. and 95° C. The use of PCR to amplify DNA or RNA has greatly advanced a wide range of disciplines, ranging from basic biology to clinical diagnostics and forensics. One particular form of PCR that is often used in diagnostics and biomedical research is quantitative PCR (qPCR), which not only detects the presence of DNA or RNA in the sample, but also provides an accurate measure of its concentration. This is an important data point for making subsequent decisions and analysis—for example, the amount of a HIV therapeutic drug, that is given to the patient, is determined by the amount of detected viral RNA load in the test sample.
So far, the most common form to carry out qPCR is real-time PCR, which is widely used in many areas, including basic biomedical research and clinical diagnostics. In real-time PCR, the absolute concentration of a sample is inferred from the time evolution of the amplification process, which is monitored with a fluorescent probe, such as a molecular beacon or Taqman® probe, that specifically recognizes the amplification product. Real-time PCR is susceptible to various errors, including the formation of unwanted primer dimers, where primer molecules attach to each other because of complementary stretches in their sequence. As a result, a by-product is generated which competes with the target element for available PCR reagents, thus potentially inhibiting amplification of the target sequence and interfering with accurate quantification. The quantification of target also requires the precise knowledge of the amplification efficiency for each cycle, and because the growth is exponential, tiny uncertainties in amplification efficiency (e.g. below the threshold detection level) will result in very large errors in the determination of target copy numbers. This error can become very large when the initial concentration of nucleic acid is low or when the fluorescent detection is not sufficiently sensitive. Despite its power to identify and quantify target DNA from complex samples, real time PCR suffers from the inability to quantify low sample concentrations with sufficient precision, as required for example in the detection of pathogens or clinical diagnostics.
To overcome the difficulties of real-time PCR to quantify low copy-number DNA, digital or limiting dilution DNA amplification has been developed, which can quantify the absolute number of template copies in the sample more accurately. In dPCR, the total sample is divided into an array of small volumes, such that, based on Poisson statistics, only few volumes contain one or more target molecules, while the majority of volumes contains no DNA. DNA amplification is then carried out in all volumes simultaneously and results in an increase of fluorescence in only those few volumes that contain target molecules. The DNA copy number is easily and accurately determined by counting the number of fluorescent volumes (i.e. those that contain a copy of DNA).
The concept of dPCR is appealing, but it is not yet widely used because (1) it can be difficult to create a large array of very small volumes (picoliters to nanoliters) used for dPCR, and (2) the dynamic range of the experiment is defined by the size and number of discrete arrays and is often very low. In order to accurately quantify the amount of DNA or protein in the sample most of the compartments typically contain at maximum one target molecule. This implies that the initial concentration of sample be matched to the dynamic range of the assay. In other words, the initial sample concentration should be determined before inputting the correct concentration of sample into the device to run dPCR. This adds to the inconvenience of running dPCR and limits the potential of digital arrays with constant volumes.
Regardless of the particular reaction used, it is important to overcome the limited dynamic range of the digital assay. A straightforward way is to extend the scope of the assay by increasing the number of digitized volumes of the same size. This approach is problematic; in order to accommodate a large number of volumes, the device has to be large and would involve fairly complex and expensive microfabrication. Thus, there is a need for additional methods and systems for performing digital measurements.
Besides the above mentioned practical issues of dPCR, widespread use of the method can also be impeded by precise temperature control and temperature cycling. Generally, the temperature for the annealing and melting step is controlled within +/−1 degree Celsius. For many applications, where absolute quantification of DNA and RNA is important, these factors are difficult to meet or expensive to realize, in particular in resource-limited settings and at the point-of-care. To provide more ergonomic ways to amplify DNA and RNA in these settings, several isothermal methods have been developed, including rolling circle amplification (RCA), nucleic acid sequence based amplification (NASBA) or loop-mediated amplification (LAMP).
LAMP is an isothermal process for amplifying DNA or RNA with very high specificity at a fixed temperature between 60-65 degrees Celsius. Due to its high specificity it is able to discriminate single nucleotide differences during amplification. As a result, LAMP has been applied for SNP (single nucleotide polymorphism) typing. LAMP has also been shown to detect viral RNA with about ten-fold higher sensitivity than RT-PCR. Another feature, that differentiates LAMP from other isothermal methods, is the ability to directly correlate the amplification of DNA with the production of magnesium pyrophosphate, which increases the turbidity of the solution. The progress of the LAMP solution can thus be followed with a simple turbidimeter. Therefore, a non-homogeneous assay can be used for detecting the amplification products that result from LAMP. The production of magnesium pyrophosphate can also be used in form of a fluorescent indicator, which is particularly useful for digital assay readout. Before the reaction, a small amount of Calcein is added to the reaction mix. During amplification, the increased production of pyrophosphate leads to a sharp increase in Calcein fluorescence in those volumes that contain one or more target molecules.
These reactions proceed at a fixed temperature, which reduces instrument complexity and lowers energy consumption, making them more suitable for point-of-care diagnostics and home-medicine devices. Translation of these methods into a digital format is an important step towards a better and more accurate detection of pathogens at the point-of-care. Moreover, digital assays would also improve the accuracy of protein amplification based assays, such as ELISA (Enzyme-Linked-Immunoadsorbent-Assay) or any single molecule based assay, where the single molecule assay may or may not require amplification.
In view of the above, there is a need to provide improved methods and systems for performing digital measurements. In addition, there is also a need to provide additional techniques using digital measurements, such as digital LAMP. The present invention disclosed herein provides these needs and more.